This invention relates to nucleic acid polymerization and amplification. In particular, it relates to a novel and general method for nucleic acid amplification, in which pyrophosphorolysis and polymerization are serially-coupled. The method has been adapted for allele-specific amplification and can greatly increase the specificity to detect an extremely rare allele in the presence of wild type alleles. We refer to the method as pyrophosphorolysis activated polymerization (PAP).
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended Lists of References.
A method of detecting one mutant allele in 106-109 wild type alleles would be advantageous for many applications including detecting minimal residual disease (rare remaining cancer cells during remission, especially mutations in the p53 gene or other tumor suppressor genes previously identified within the tumors) and measurement of mutation load (the frequency of specific somatic mutations present in normal tissues, such as blood or urine). Individuals with a high mutation load may be at increased risk for cancer to either environmental exposure or endogenous defects in any of hundreds of genes necessary to maintain the integrity of the genome. For those individuals found to have a high mutation load, clues to etiology can be obtained by defining the mutation pattern.
Multiple methods for detecting mutations present in less than 10% of cells (i.e. rare alleles) have been developed including PCR amplification of specific alleles (PASA), PNA clamping blocker PCR, allele specific competitive blocker PCR, MAMA, and RFLP/PCR (1). These methods: i) amplify the rare allele selectively, ii) destroy the abundant wild type allele, or iii) spatially separate the rare allele from the wild type allele. RFLP/PCR has been reported to have the highest specificity of 10xe2x88x928 (2), but in our hands the specificity has been 10xe2x88x923 to 10xe2x88x924 (3). Methods that selectively amplify the rare allele include PASA, which routinely has a specificity of less than or equal to 1 part in 40 (4).
DNA polymerases, which are critical to DNA amplification, catalyze some or all of the following reactions: i) polymerization of deoxynucleotide triphosphates; ii) pyrophosphorolysis of duplexes of DNA in the presence of pyrophosphate (PPi); iii) 3xe2x80x2-5xe2x80x2 exonuclease activity and iv) 5xe2x80x2-3xe2x80x2 exonuclease activity (5, 6). For Taq and Tfl DNA polymerases, the polymerization and 5xe2x80x2-3xe2x80x2 exonuclease activity have been reported (7-9). For T7 Sequenase(trademark) DNA polymerases, pyrophosphorolysis can lead to the degradation of specific dideoxynucleotide-terminated segments in Sanger sequencing reaction (10, 11).
There are many DNA sequencing methods and their variants, such as the Sanger sequencing using dideoxy termination and denaturing gel electrophoresis (27), Maxam-Gilber sequencing using chemical cleavage and denaturing gel electrophoresis (28), pyro-sequencing detection pyrophosphate (PPi) released during the DNA polymerase reaction (29), and sequencing by hybridization (SBH) using oligonucleotides (30-35).
Herein, we describe pyrophosphorolysis activated polymerization (PAP), an approach which has the potential to enhance dramatically the specificity of PASA. We also describe a novel method of DNA sequence determination by PAP.
The invention is a pyrophosphorolysis activated polymerization (PAP) method of synthesizing a desired nucleic acid strand on a nucleic acid template strand. The method comprises the following steps carried out serially.
(a) Annealing to the template strand a complementary activatable oligonucleotide P*. This activatable oligonucleotide has a non-extendable 3xe2x80x2-deoxynucleotide at its 3xe2x80x2 terminus. It has no nucleotides at or near its 3xe2x80x2 terminus that mismatch the corresponding nucleotides on the template strand. Therefore, the terminal 3xe2x80x2-deoxynucleotide is hybridized to the template strand when the oligonucleotide P* is annealed.
(b) Pyrophosphorolyzing the annealed activatable oligonucleotide P* with pyrophosphate and an enzyme that has pyrophosphorolysis activity. This activates the oligonucleotide P* by removal of the hybridized terminal 3xe2x80x2-deoxynucleotide.
(c) Polymerizing by extending the activated oligonucleotide P* on the template strand in presence of four nucleoside triphosphates and a nucleic acid polymerase to synthesize the desired nucleic acid strand.
The PAP method can be applied to amplify a desired nucleic acid strand by the following additional steps.
(d) Separating the desired nucleic acid strand of step (c) from the template strand, and
(e) Repeating steps (a)-(d) until a desired level of amplification of the desired nucleic acid strand is achieved.
In a preferred aspect, the PAP method as described above is applied to allele-specific amplification. In this application, the nucleic acid template strand is a sense or antisense strand of one allele and is present in admixture with the corresponding (sense or antisense) nucleic acid strand of the second allele (the allelelic strand). The activatable oligonucleotide P* has at least one nucleotide at or near its 3xe2x80x2 terminus that mismatches the corresponding nucleotide of the allelic strand. Because of the mismatch, in step (a) of the PAP method the terminal 3xe2x80x2-deoxynucleotide of oligonucleotide P* is not hybridized to the allelelic strand. In step (b) the pyrophosphorolysis does not substantially remove the non-hybridized terminal 3xe2x80x2-deoxynucleotide from the activatable oligonucleotide P* annealed to the allelic strand. In step (c) the oligonucleotide P* is not substantially extended by polymerization on the allelic strand. As a result, the desired nucleic acid strand synthesized on the template strand is amplified preferentially over any nucleic acid strand synthesized on the allelelic strand.
The PAP method can be used to amplify either RNA or DNA. When used to amplify DNA, the activatable oligonucleotide P* is a 2xe2x80x2-deoxyoligonucleotide, the terminal deoxynucleotide is a 2xe2x80x2,3xe2x80x2-dideoxynucleotide, the four nucleoside triphosphates are 2xe2x80x2-deoxynucleoside triphosphates, and the nucleic acid polymerase is a DNA polymerase. The DNA polymerase used in step (c) can also be the enzyme having pyrophosphorolysis activity used in step (b). Preferred DNA polymerases having pyrophosphorolysis activity are thermostable Tfl, Taq, and genetically engineered DNA polymerases, such as AmpliTaqFs and ThermoSequenase(trademark). These genetically engineered DNA polymerases have the mutation F667Y in their active sites and elimination of 5xe2x80x2-3xe2x80x2 exonuclease activity. The use of genetically engineered DNA polymerases, such as AmpliTaqFs and ThermoSequenase(trademark), greatly improves the efficiency of PAP.
Amplification by the PAP method can be linear or exponential. Linear amplification is obtained when the activatable oligonucleotide P* is the only complementary oligonucleotide used. Exponential amplification is obtained when a second oligonucleotide is present that is complementary to the desired nucleic acid strand. The activatable oligonucleotide P* and the second oligonucleotide flank the region that is targeted for amplification. In step (a) the second oligonucleotide anneals to the separated desired nucleic acid strand product of step (d). In step (c) polymerization extends the second oligonucleotide on the desired nucleic acid strand to synthesize a copy of the nucleic acid template strand. In step (d) the synthesized nucleic acid template strand is separated from the desired nucleic acid strand. Steps (a) through (d) are repeated until the desired level exponential amplification has been achieved.
In the PAP method, a mismatch between the activatable oligonucleotide P* and the template strand results in no amplification, if the mismatch occurs in the 3xe2x80x2 specific subsequence of P* at the 3xe2x80x2 terminus of P* or within 16 nucleotides of the 3xe2x80x2 terminus of P*. This lack of amplification for such mismatches in the 3xe2x80x2 specific subsequence of P* provides four billion different and specific oligonucleotides with one base substitution resolution.
In a preferred aspect, the PAP method is used for exponential amplification of a rare, mutant allele in a mixture containing one or more wild-type alleles. Strands of the alleles are separated to provide single-stranded DNA, then the following steps are carried out serially.
(a) Annealing to the sense or antisense strands of each allele a complementary activatable 2xe2x80x2-deoxyoligonucleotide P* that has a non-extendable 2xe2x80x2,3xe2x80x2-deoxynucleotide at its 3xe2x80x2 terminus. P* has no 2xe2x80x2-deoxynucleotides at or near its 3xe2x80x2 terminus that mismatch the corresponding 2xe2x80x2-deoxynucleotides on the mutant strand, but has at least one 2xe2x80x2-deoxynucleotide at or near its 3xe2x80x2 terminus that mismatches the corresponding 2xe2x80x2-deoxynucleotide on the wild type stand. Consequently, the terminal 2xe2x80x2,3xe2x80x2-deoxynucleotide is hybridized to the mutant strand but not to the wild-type strand when the oligonucleotide P* is annealed. Simultaneously, a second 2xe2x80x2-deoxyoligonucleotide that is complementary to the anti-parallel strands of each allele is annealed to the anti-parallel strands. The activatable 2xe2x80x2-deoxyoligonucleotide P* and the second 2xe2x80x2-deoxyoligonucleotide flank the region of the gene to be amplified.
(b) Pyrophosphorolyzing the activatable 2xe2x80x2-deoxyoligonucleotide P* that is annealed to a mutant strand with pyrophosphate and an enzyme that has pyrophosphorolysis activity. This activates the 2xe2x80x2-deoxyoligonucleotide P* that is annealed to the mutant strand by removal of the hybridized terminal 2xe2x80x2,3xe2x80x2-deoxynucleotide. It does not substantially activate the 2xe2x80x2-deoxyoligonucleotide P* that is annealed to the mutant strand because the non-hybridized terminal 2xe2x80x2,3xe2x80x2-deoxynucleotide is not substantially removed by the pyrophosporolysis.
(c) Polymerizing by extending the activated oligonucleotide P* on the mutant strand in presence of four nucleoside triphosphates and a DNA polymerase and simultaneously extending the second 2xe2x80x2-deoxyoligonucleotide on both mutant and wild-type anti-parallel strands.
(d) Separating the extension products of step (c);
(e) Repeating steps (a)-(d) until the desired level of exponential amplification of the mutant allele has been achieved.
The activatable 2xe2x80x2-deoxyoligonucleotide P* is annealed to the antisense strands of the alleles and the second 2xe2x80x2-deoxyoligonucleotide is annealed to the sense strands, or vice versa.
Steps (a) to (c) of PAP can be conducted sequentially as two or more temperature stages on a thermocycler, or they can be conducted as one temperature stage on a thermocycler.
Nucleoside triphosphates and 2xe2x80x2-deoxynucleoside triphosphates or their chemically modified versions may be used as substrates for multiple-nucleotide extension by PAP, i.e., when one nucleotide is incorporated the extending strand can be further extended. 2xe2x80x2,3xe2x80x2-dideoxynucleoside triphosphates or their chemically modified versions which are terminators for further extension may be used for single-nucleotide extension. 2xe2x80x2,3xe2x80x2 dideoxynucleoside triphosphates may be labeled with radioactivity or fluorescence dye for differentiation from the 3xe2x80x2 terminal dideoxynucleotide of oligonucleotide P*. Mixtures of nucleoside triphosphates or 2xe2x80x2-deoxynucleotide triphosphates and 2xe2x80x2,3xe2x80x2-dideoxynucleoside triphosphates may also be used.
PAP can be used in a novel method of DNA sequence determination. In PAP, pyrophosphorolysis and polymerization by DNA polymerase are coupled serially by using P*, a 3xe2x80x2 dideoxy terminal oligonucleotide. This principle is based on the specificity of PAP and in turn on the base pairing specificity of the 3xe2x80x2 specific subsequence. This property of the 3xe2x80x2 specific subsequence can be applied to scan for unknown sequence variants, to determine de novo DNA sequence, to compare two DNA sequences, and to monitor gene expression profiling in large scale. A P* array is possible in these methods. That is, each of the P*s can be immobilized at an individual dot or a two dimensional solid support, thus allowing all the PAP reactions to be processed in parallel.
Thus in one aspect, the PAP method is used for scanning unknown sequence variants in a nucleic acid sequence or for resequencing of a predetermined sequence in a nucleic acid by carrying out the following steps serially.
(a) Mixing under hybridization conditions a template strand of the nucleic acid with multiple sets of four activatable oligonucleotides P* which are sufficiently complementary to the template strand to hybridize therewith. Within each set the oligonucleotides P* differ, from each other in having a different 3xe2x80x2-terminal non-extendable nucleotide, so that the 3xe2x80x2 terminal non-extendable nucleotide is hybridized to the template strand if the template strand is complementary to the 3xe2x80x2 terminal non-extendable nucleotide. The number of sets correspond to the number of nucleotides in the sequence.
(b) Treating the resulting duplexes with pyrophosphate and an enzyme that has pyrophosphorolysis activity to activate by pyrophosphorolysis only those oligonucleotides P* which have a 3xe2x80x2 terminal non-extendable nucleotide that is hybridized to the template strand.
(c) Polymerizing by extending the activated oligonucleotides P* on the template strand in presence of four nucleoside triphosphates and a nucleic acid polymerase.
(d) Separating the nucleic acid strands synthesized in step (c) from the template strand.
(e) Repeating steps (a)-(d) until a desired level of amplification is achieved, and
(f) Arranging the nucleic acid sequence in order by analyzing overlaps of oligonuclotides P* that produced amplifications.
In a second aspect, the PAP method is used for determining de novo the sequence of a nucleic acid by carrying out the following steps serially.
(a) Mixing under hybridization conditions a template strand of the nucleic acid with multiple activatable oligonucleotides P*. All of the oligonucleotides P* have the same number n of nucleotides as the template and constitute collectively all possible sequences having n nucleotides. All of the oligonucleotides P* have a non-extendable nucleotide at the 3xe2x80x2 terminus. Any oligonucleotides P* that are sufficiently complementary will hybridize to the template strand. The 3xe2x80x2 terminal non-extendable nucleotide will hybridize to the template strand only if the template strand is complementary at the position corresponding to the 3xe2x80x2 terminus.
(b) Treating the resulting duplexes with pyrophosphate and an enzyme that has pyrophosphorolysis activity to activate only those hybridized oligonucleotides P* which have a 3xe2x80x2 terminal non-extendable nucleotide that is hybridized to the template strand, by pyrophosphorolysis of those hybridized 3xe2x80x2 terminal non-extendable nucleotides.
(c) Polymerizing by extending the activated oligonucleotides P* on the template strand in presence of four nucleoside triphosphates and a nucleic acid polymerase.
(d) Separating the nucleic acid strands synthesized in step (c) from the template strand.
(e) Repeating steps (a)-(d) until a desired level of amplification has been achieved, and
(f) Determining the sequence of oligonucleotides P* that produced amplifications, then arranging the nucleic acid sequence in order by analyzing overlaps of these oligonucleotides.